Department of Molecular Biology, Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic.

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Commonwealth Scientific and Industrial Research Organization (CSIRO), Food and Nutrition Flagship, North Ryde, New South Wales, Australia.

Abstract

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. Here, we show that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology.

(A) A luciferase reporter construct carrying 8 copies of a JH response element (JHRE). The JHRE and the basal promoter derive from the Aedes aegypti early trypsin gene [,]. The core (capital letters) of the JHRE was mutated to produce mutJHRE. (B) S2 cells responded to the indicated compounds by activating the JHRE-luc reporter, but not mutJHRE-luc (1 μM JH III). Farnesol is a biologically inactive control. (C-D) Activation of JHRE-luc by 1 μM JH III or 10 μM MF relative to basal activity (ethanol) required both Gce and Tai as revealed by dsRNA-mediated knockdown of either protein; egfp dsRNA served as a control. Co-transfection with a Tai-expressing plasmid enhanced the Gce-dependent activation by JH III and MF (D). Data were normalized to Renilla luciferase activity and plotted as mean ± SD (n = 3).

Gce and Met mutated in their JH-binding domains are incapable of activating transcription.

(A) Positions of three conserved amino acids important for binding of JH III based on our model of the T. castaneum Met PAS-B domain []. (B) Only wild-type Gce (WT) capable of binding JH III () activated the JHRE-luc reporter in S2 cells. (C) Similar results were obtained for Met, which also lost its ability to activate JHRE-luc in response to JH III when its PAS-B domain was mutated at the corresponding conserved residues. In both experiments (B and C), the endogenous gce and Met were suppressed by RNAi. Data were normalized to Renilla luciferase activity and plotted as mean ± SD (n = 3). The WT and mutated Gce and Met variants were all stable as detected on immunoblots (insets) using their FLAG tags; antibody against the Mbf1 or Cheerio proteins served as controls.

The ligand-binding capacity of Gce is required for normal expression of the direct JH-response gene Kr-h1 in vivo.

Reduction in Kr-h1 mRNA levels in Met27gce2.5k mutant larvae can be compensated by transgenic expression of the functional Gce protein but not by any of its mutated forms, incapable of binding JH III (). Balanced Met27gce2.5k/FM7c; arm-Gal4 females were crossed with males bearing the UAS-gce transgenes. The male progeny were selected as mid-third instar larvae and genotyped by PCR detecting the gce2.5k deletion [] to distinguish Met27gce2.5k/Y from Met+gce+/Y siblings carrying the FM7c balancer chromosome. Groups of four larvae were subjected to qRT-PCR with primers detecting the endogenous Kr-h1α and gce transcripts (). Values relative to mRNA levels in Met+gce+/Y controls (set to 1) are mean ± SD from three biological replicates for each genotype, five replicates for GceWT. The significance levels of differences determined by t-test were P < 0.02 (*), P = 0.0002 (**), and P < 0.0002 (***).

(A-B) Met27 mutants tolerate methoprene better than Met+ control flies [] or Met27 flies expressing transgenic GceWT or MetWT proteins. Met27/Y males carrying the indicated UAS-gce or UAS-Met transgenes were mated to Met27; arm-Gal4 females, and the F1 progeny was fed methoprene. In the presence of methoprene, GceWT totally blocked adult development and MetWT significantly (*; P < 0.0003) reduced survival relative to the Met27 strain, whereas mutated Gce or Met did not have this effect. Values are per cent average numbers of emerged adults relative to total numbers of pupated animals. Each column represents 200–430 animals (or the indicated numbers in B) counted in 2–3 independent trials. (C-D) Balanced Met27gce2.5k/FM7c; arm-Gal4 (or tub-Gal4) females were crossed with males bearing the UAS-gce or UAS-Met transgenes, and emerged Met27gce2.5k/Y adult males were scored relative to their FM7c/Y (Met+gce+) siblings (1:1 ratio was considered 100% rescue). Non-conditionally lethal Met27gce2.5k/Y males were rescued to adulthood by transgenic GceWT, FLAG-GceWT, and MetWT proteins but not by their mutated versions except for a few flies rescued by one randomly inserted FLAG-GceV315F construct expressed under arm-Gal4 (C). Data are mean ± SD; numbers of all scored F1 males are given above columns. (E) FLAG-Gce WT and mutated proteins were stable when expressed in vivo as shown on an immunoblot (anti-FLAG antibody) from adult flies. The Cheerio (Cher) protein served as a control. (F) FLAG-Gce WT and mutated proteins were detected with the anti-FLAG antibody in the nuclei of larval fat body cells. Bars, 50 μm.